CDX2 Expression and Fluoropyrimidine Response in Rare Non-GI Tumors: A Three-Case Series

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors reported a retrospective case series of three patients with metastatic adenocarcinoma (aggressive variant prostate, minor salivary gland, and intestinal-type sinonasal tract) exhibiting strong CDX2 nuclear expression and were refractory to or lacked established standard systemic therapy. The patients were treated with fluoropyrimidine-based regimens based on the strong CDX2 expression in tumor cells and all three patients achieved marked metabolic and clinical responses. These findings are interesting and the manuscript is well written. I have one question:

I am wondering if there could be a selection bias. How many similar patients with CDX2 expressing metastatic adenocarcinoma were treated during this period? Since all three patients in this series had excellent response, it gives an impression that the reponse rate is 100% which might not be true. The authors acknowledged the limitation in discussion but it probably should be acknowleged in the abstract as well.

Author Response

Comment1: The authors reported a retrospective case series of three patients with metastatic adenocarcinoma (aggressive variant prostate, minor salivary gland, and intestinal-type sinonasal tract) exhibiting strong CDX2 nuclear expression and were refractory to or lacked established standard systemic therapy. The patients were treated with fluoropyrimidine-based regimens based on the strong CDX2 expression in tumor cells and all three patients achieved marked metabolic and clinical responses. These findings are interesting and the manuscript is well written. I have one question:

I am wondering if there could be a selection bias. How many similar patients with CDX2 expressing metastatic adenocarcinoma were treated during this period? Since all three patients in this series had excellent response, it gives an impression that the reponse rate is 100% which might not be true. The authors acknowledged the limitation in discussion but it probably should be acknowleged in the abstract as well.

Response: We thank Reviewer 1 for this insightful comment regarding potential selection bias and interpretation of response rates. The three patients included in this report represent consecutive cases identified during the study period with metastatic adenocarcinoma of non-colorectal origin demonstrating strong nuclear CDX2 expression who were treated with fluoropyrimidine-based therapy. No additional patients meeting these criteria were treated during this time frame; therefore, the denominator is three.

We agree that the small sample size and case series methodology may give the impression of a 100% response rate and preclude inference regarding treatment efficacy. To address this concern, we have revised both the Abstract and Discussion to explicitly clarify that this report is descriptive and hypothesis-generating and not intended to estimate response rates or treatment efficacy.

Revised text added to the Abstract (marked in red in the manuscript):
“This report is hypothesis-generating and not intended to estimate response rates or treatment efficacy.”

Corresponding revisions were also made in the Discussion (Limitations section) (marked in red in the manuscript).

"Consequently, this report is descriptive and hypothesis-generating and should not be interpreted as evidence of 100% response rate or treatment efficacy."

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

it is absolutely necessary to provide figures for the immunohistochemical stainings you report in your manuscript!!

Furthermore in case 2 you say: Evaluation in the Head and Neck Oncology Clinic at Mayo Clinic favored a diagnosis of minor salivary gland adenocarcinoma with enteric differentiation and regional nodal metastases. Surgical resection would have required near total glossectomy, severely impacting quality of life; therefore, non-surgical management was prioritized in alignment with the patient’s preference for organ preservation.

Was that the reason, or the fact that the prognosis was poor and the glossectomy would probably make no difference in the clinical outcome? 

Is there any intraoral picture/ documentation for the possibility of minor salivary involvement? Was an intraoral biopsy performed? 

In any case, the major issue is the lack of immunohistochemistry. You have to provide the evidence that support the diagnosis so that we may be able to review it as well.

Author Response

Comment1: it is absolutely necessary to provide figures for the immunohistochemical stainings you report in your manuscript!!

Answer: We thank Reviewer 2 for this comment. High-resolution CDX2 immunohistochemistry images have been added for all three cases (Figure 1), demonstrating nuclear CDX2 expression consistent with the reported diagnoses. Representative low- and high-power images are provided. All slides were reviewed by board-certified pathologists at Mayo Clinic.

Comment2: Furthermore in case 2 you say: Evaluation in the Head and Neck Oncology Clinic at Mayo Clinic favored a diagnosis of minor salivary gland adenocarcinoma with enteric differentiation and regional nodal metastases. Surgical resection would have required near total glossectomy, severely impacting quality of life; therefore, non-surgical management was prioritized in alignment with the patient’s preference for organ preservation.

Was that the reason, or the fact that the prognosis was poor and the glossectomy would probably make no difference in the clinical outcome? 

Response: We thank Reviewer 2 for raising this important point. In head and neck malignancies, induction chemotherapy may be used in select cases to reduce tumor burden and facilitate organ preservation, although it is not routinely employed due to modest historical response rates, particularly in biologically heterogeneous salivary gland malignancies.

In this case, definitive surgical management with curative intent would have required near-total glossectomy followed by high-dose adjuvant radiotherapy. While technically feasible, this approach would have resulted in substantial and permanent functional morbidity, including severe impairment of speech and swallowing. In addition, extensive disease would have necessitated large radiation fields involving critical structures, increasing the risk of long-term dysphagia and fibrosis.

Given the tumor’s strong nuclear CDX2 expression, suggesting activation of an intestinal differentiation program associated with fluoropyrimidine sensitivity, induction fluoropyrimidine-based chemotherapy was pursued with curative intent to reduce tumor burden and facilitate organ preservation prior to definitive radiotherapy. We have clarified this rationale in the revised Case 2 description.

Revised text added to the Case 2 description (marked in red in the manuscript):

Definitive surgical management with curative intent would have required near-total glossectomy followed by high-dose adjuvant radiotherapy, resulting in substantial and permanent functional morbidity, including severe impairment of speech and swallowing. Although technically feasible, this approach was associated with a high risk of long-term functional decline. Therefore, non-surgical management was prioritized to preserve organ function, in alignment with the patient’s preference for organ preservation.

Comment3: Is there any intraoral picture/ documentation for the possibility of minor salivary involvement? Was an intraoral biopsy performed? 

Response: We thank Reviewer 2 for this comment. No intraoral photographs are available. The diagnosis was based on biopsy of a cervical lymph node, comprehensive histopathologic and immunohistochemical review, and the anatomic distribution of disease. A direct intraoral biopsy was not obtained. We have added clarification regarding how the diagnosis was established to the Case 2 description.

Revised text added to the Case 2 description (marked in red in the manuscript):

"Evaluation in the Head and Neck Oncology Clinic at Mayo Clinic favored a diagnosis of minor salivary gland adenocarcinoma with enteric differentiation and regional nodal metastases, based on histopathologic review of the available tissue and the anatomic distribution of disease, as a direct intraoral biopsy was not obtained".

Comment4: In any case, the major issue is the lack of immunohistochemistry. You have to provide the evidence that support the diagnosis so that we may be able to review it as well.

Response: We agree with the reviewer regarding the importance of immunohistochemical documentation. While original pathology slides cannot be shared for external review, representative high-quality CDX2 immunohistochemistry images have been added to the revised manuscript (Figure 1), along with corresponding histologic context. All cases were reviewed by board-certified pathologists at Mayo Clinic, and the provided images support the reported diagnoses.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors applied CDX2 as a biomarker in the treatment and monitoring of rare non-GI tumors. Three cases were included. Below are a number of issues that the authors shall address or revise:

1. In this study, the expression level of CDX2 was important. The authors should also provide IHC results or other results that could reflect the expression level of CDX2 in each patient.
2. As a retrospective case report without a control group, this study cannot exclude the influence of confounding factors on the observed treatment responses. The authors could give more discussion on this issue.
3. It would be valuable to include an evaluation of the diagnostic performance of CDX2, specifically addressing its sensitivity and specificity as a biomarker in this context.

 

Author Response

Comment1: In this study, the expression level of CDX2 was important. The authors should also provide IHC results or other results that could reflect the expression level of CDX2 in each patient

Response: We thank Reviewer 3 for this comment. We have added high-power CDX2 immunohistochemistry images for all three cases (Figure 1) and included detailed staining results in the text, reflecting staining intensity and the percentage of tumor cells demonstrating nuclear CDX2 expression.

Revised text added to the Cases description (marked in red in the manuscript):

Case1: with patchy strong nuclear staining for CDX2.

Case2: diffuse strong nuclear positivity for CDX2.

Case3: and diffuse strong nuclear CDX2

Comment2: As a retrospective case report without a control group, this study cannot exclude the influence of confounding factors on the observed treatment responses. The authors could give more discussion on this issue.

Response: We agree with Reviewer 3 that confounding factors may have contributed to the observed treatment responses. We have revised the Discussion and Limitations sections to explicitly acknowledge that, as a small retrospective case series without a control group, patient-specific factors, prior therapies, and tumor biology beyond CDX2 status may have influenced clinical outcomes.

Revised text added to the Discussion (Limitations section) (marked in red in the manuscript):

The favorable outcomes observed may reflect chance findings, selection bias, patient-specific factors, prior treatments, or other tumor- and patient-specific biological features beyond CDX2 status alone.

Second, we did not systematically evaluate all CDX2-positive tumors during the study period, nor did we include control comparisons with CDX2-negative tumors or CDX2-positive tumors treated with alternative regimens.

Comment3: It would be valuable to include an evaluation of the diagnostic performance of CDX2, specifically addressing its sensitivity and specificity as a biomarker in this context.

Response: We thank Reviewer 3 for highlighting this important point. While formal evaluation of CDX2 sensitivity and specificity cannot be determined from this small case series, we have added a statement referencing our larger retrospective cohort of 209 CDX2-positive patients, in which the overall response rate to fluoropyrimidine-based therapy was approximately 70%. This provides additional context for the potential clinical relevance of CDX2 expression while emphasizing that these findings remain hypothesis-generating and require prospective validation.

Revised text added to the Discussion (marked in red in the manuscript):

Although formal evaluation of CDX2 sensitivity and specificity in predicting fluoropyrimidine response cannot be determined from this small series, our prior retrospective cohort of 209 CDX2-positive cancers of unknown primary demonstrated an overall response rate to fluoropyrimidine-based therapy of approximately 70%, supporting the potential biological relevance of CDX2 expression in guiding therapy. These findings remain hypothesis-generating and warrant prospective validation.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Very nice